This application is filed pursuant to 35 U.S.C. xc2xa7371 as a United States National Phase Application of International Application No. PCT/EP98/08085 filed Dec. 16, 1998, which claims priority from GB 9726630.8 filed Dec. 18, 1997.
The present invention relates to methods for treating diabetes mellitus. The present invention also relates to antagonists of the potassium channel Kv2.1, methods for using and preparing the antagonists, and assays for identifying such antagonists.
The secretion of insulin by the pancreatic xcex2-cell plays a critical role in regulating glucose metabolism. Derangement in insulin secretion can therefore lead to impaired regulation of blood glucose, manifesting as hypoglycemia or hyperglycemia resulting in for example insulin-dependent (Type 1) diabetes mellitus or non-insulin dependent (Type 2) diabetes mellitus (NIDDM). Unlike type 1 diabetes, with NIDDM there is no autoimmune destruction of pancreatic xcex2-cells.
The treatment of Type 2 or non-insulin dependent diabetes mellitus (NIDDM) remains unsatisfactory despite the widespread use of insulin and oral agents (sulfonylureas, biguanides and thiazolidinediones). Unfortunately the available oral agents suffer from a number of undesirable side effects which limit their usefulness in treatment of NIDDM. There is thus a clear need for the development of novel hypoglycaemic agents which may be less toxic or which suceed where others are ineffective.
xcex2-cells located within the islets of Langerhans in the pancreas respond to an increase in glucose concentration by secreting insulin (Boyd A E III, J. Cell Biochem., 48, 234-241, 1992). The mechanism by which glucose acts as an insulin secretagogue is not completely understood, but a major component of its effect on xcex2-cells is mediated via changes in K+ conductance. A number of types of potassium channels are present in xcex2-cells: these include calcium activated potassium channels, ATP sensitive potassium channels (KATP), and delayed rectifier potassium channels (Kv) (Dukes I D, Philipson L H, Diabetes, 45, 845-853, 1996). The respective roles of these potassium channels in regulating glucosexe2x80x94stimulated insulin secretion has not been fully elucidated.
Kv channels are multimeric membrane proteins that permit the efflux of K+ from cells when the membrane potential is excited (i.e. depolarized) (Hille, 1993 Ionic Channels of Excitable Membranes, 2nd ed., Sunderland M A, Sinauer Associates, 1991). A number of isoforms of Kv channels have been described, and a uniform nomenclature for their naming has been agreed (Chandy K G, Gutman G A, Trends Pharmacol. Sci., 14, 434, 1993). There are currently 8 families of mammalian delayed rectifier potassium channel genes (Kv1.xxe2x88x928.x), 4 of which (Kv1.x4.x) have been demonstrated to encode functional ion channels (Chandy K G, Gutman G A in Ligand and Voltage Gated Ion Channels, Ed. North R A, CRC Press, 1994). The information relating to Kv channel gene expression in pancreatic xcex2-cells is somewhat contradictory. Whereas some groups, using polymerase chain reaction (PCR), have detected Kv1.x isoforms in mouse islets and insulinoma cells (Betshlolz C, et al, FEBS Lett, 263, 121-123, 1990; Kalman K, et al, Biophys J 68:A268, 1995), and U.S. Pat. No. 5,559,009 discloses the existence of Kv1.7 message in rat pancreatic xcex2 cells and hamster insulinoma cells, others have failed to detect expression of these isoforms in xcex2-cells, instead reporting the expression of Kv2.1 and 3.2 transcripts (Roe M W, et al, J Biol Chem 271, 32241-32246, 1996).
The present inventors have surprisingly found that antagonists of the delayed rectifier potassium channel Kv2.1 enhance insulin secretion. Accordingly, antagonists of the delayed rectifier potassium channel Kv2.1 are useful in the treatment of NIDDM.
Briefly, in one aspect, the present invention provides a method of treating NIDDM in a subject, comprising administering to the subject a therapeutically effective amount of an antagonist of the delayed rectifier potassium channel Kv2.1.
In another aspect, the present invention provides the novel compound of Formula (1), or a pharmaceutically acceptable salt or solvate thereof 
wherein R represents C1-12alkyl, C1-12alkenyl, xe2x80x94CyH2yxe2x80x94R7 or xe2x80x94CyH2yxe2x80x94Oxe2x80x94R7 where y is an integer from 1-6 and R7 is hydrogen, C1-6alkyl, C3-7cycloalkyl, C4-7cycloalkenyl, or a C5-6heterocyclic group, or R represents xe2x80x94CyH2yxe2x80x94R9, xe2x80x94CyH2yxe2x80x94Oxe2x80x94R9, or xe2x80x94CyH2yxe2x80x94Oxe2x80x94CH2xe2x80x94R9, where y is independently as defined above and R9 is 
where R4 is hydrogen or halogen, and R5 and R6 independently represent hydrogen, halogen, xe2x80x94Oxe2x80x94C1-3alkyl, or R5 and R6 can together form a methylenedioxy or ethylenedioxy ring;
R1 represents 
xe2x80x83where each R8 is independently hydrogen, halogen, xe2x80x94Oxe2x80x94C1-3alkyl, C1-3alkyl, C1-3fluoroalkyl, or xe2x80x94C(O)xe2x80x94R9 where R9 is C4-8alkyl, or C3-7cycloalkyl;
X represents O, S, or Nxe2x80x94R2 where R2 is independently as defined above for R;
R3 represents H, or C1-3 alkyl.
As used herein terms such as alkyl, alkenyl, and the like, can be either straight chain and branched chain unless otherwise indicated.
The compounds of formula (I) are antagonists of the delayed rectifier potassium channel Kv2.1.
The compounds of the invention are useful, for example, for the treatment of NIDDM.
In another aspect, the present invention provides the use of hanatoxin or a pharmaceutically acceptable salt or solvate thereof for the treatment of NIDDM.
In another aspect, the present invention provides a method (an assay) to identify extrinsic materials possessing the ability to modulate Kv2.1 channel activity and thereby modify insulin secretion.
The following are further particular aspects of the present invention:
a) Use of a Kv2.1 antagonist for the manufacture of a medicament for the treatment of NIDDM.
b) A compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof for use in therapy.
c) Use of a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for the treatment of NIDDM.
d) A method of treating NIDDM in a subject which comprises administering to the subject a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof.
e) A pharmaceutical composition comprising a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof together with a pharmaceutically acceptable diluent or carrier.
A number of toxins isolated from venomous animals are known to block potassium channels (Miller C, et al, Nature, 313, 316-318, 1985; Garcia-Calvo M, et al, J. Biol. Chem., 268, 18866-18874, 1993; Halliwell J V, et al, Proc. Natl. Acad. Sci. USA, 83, 493-497, 1986). Hanatoxin, isolated from the venom of the Chile Rose Tarantula (Grammulosa spatulata), has been shown to selectively block Kv2.1 channels, with minimal reported effects on representative members of Kv1, Kv3 and Kv4 delayed rectifier channels (Swartz K J, MacKinnon R, Neuron, 15, 941-949, 1995). Accordingly, hanatoxin (found in venom from the Chile Rose Tarantuala) is useful in the present invention as an antagonist of the Kv2.1 channel.
In addition to natural products, small organic molecules are known to block potassium channels. Sulfonlyureas specifically interact with KATP channels. Tetraethylammonium (TEA) and 4-aminopyridine block a wide variety of potassium channels, including those of the delayed rectifier type in the mM concentration range (Chandy K G, Gutman G A in Ligand and Voltage Gated Ion Channels, Ed. North R A, CRC Press, 1994). More potent potassium channel antagonists, have also been described. For instance, tedisamil (3,7-di(cyclopropylmethyl)-9,9-tetramethylene-3,7-diazabicyclo-(3.3.1)nonane) blocks Kv channels in the low xcexcM range with no effect on inward rectifier potassium channels. Tedisamil also has effects on voltage activated sodium and calium channels at higher concentrations (Dukes I D, et al, J. Pharmacol. Exp. Ther., 254, 560-569, 1990). 3,7-diazabicyclo-(3.3.1)-nonane (bispidine) compounds have become of considerable interest as potential antiarrhythmic agents. For instance U.S. Pat. No. 4,550,112 discloses a series of substituted 3,7-diazabicyclo-(3.3.1)-nonane compounds (including tedisamil) and their use in treating heart disease. U.S. Pat. No. 4,451,473 discloses another series of 3,7-diazabicyclo-(3.3.1)-nonane compounds that are useful as anti-arrhythmic agents. U.S. Pat. No. 4,451,473 includes the compound known as bisaramil (3-ethyl-7-methyl-3,7-diazobicyclo[3.3.1]non-9-yl 4-chlorobenzoate). Likewise, U.S. Pat. No. 4,959,373 discloses another series of compounds with the bispidine skeleton that are useful as anti-arrhythmic agents. None of these previous publications claim that compounds of this type are useful in the treatment of NIDDM.
The present inventors have found that compounds of formula (1) are potent antagonists of the delayed rectifier potassium channel Kv2.1, and enhances insulin secretion. Accordingly, compounds of formula (1) are useful in the treatment of NIDDM.
The term 5-or 6-membered heterocyclic group as used herein includes 5- or 6-membered substituted or unsubstituted heterocycloalkyl and heteroaryl groups, e.g. substituted or unsubstituted heteroarly groups e.g. substituted or unsubstituted imidazolidinyl, piperidyl, piperazinyl pyrrolidinyl, morpholinyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, pyrazolyl, imidazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxaolyl, oxadiazolyl, thiazolyl, thiadiazolyl, triazolyl or tetrazolyl.
The heterocyclic group may optionally be substituted by one or more of the following: halogen atoms, C1-3alkyl, C1-3alkoxy.
Preferably, when R is alkyl, R is C6-10alkyl. Preferably, when R7 is a heterocycle it is an unsubstituted heterocycle most preferably, R7 is furyl or thienyl. Preferably, y is 3, 4, or 5. Preferably two of the R8 substituents in R1 are hydrogen, and most preferably the other R8 is halogen. Preferably when R2 is alkyl, R2 is C1-2alkyl. Preferably R3 is methyl or hydrogen.
Example of preferred compounds of this invention are:
anti-3-(4-(3,4-methylenedioxyphenyl)butyl)-7-methyl-3,7-diazabicyclononan-9-ol 4-chlorobenzoate ester;
anti-3-(4-(3,4-ethylenedioxyphenyl)butyl)-7-methyl-3,7-diazabicyclononan-9-ol 4-chlorobenzoate ester;
anti-3-(3-methyl-3-benzyloxypropyl)-7-methyl-3,7-diazabicyclononan-9-ol 4-chlorobenzoate ester;
anti-3-(benzyl)-7-methyl-3,7-diazabicyclononan-9-ol 4-chlorobenzoate ester;
anti-3-(4-(3,4-methylenedioxyphenyl)butyl)-7,9-dimethyl-3,7-diazabicyclo-nonan-9-ol 4-chlorobenzoate;
3,7-di(4-(3,4-methylenedioxyphenyl)butyl)-3,7-diazabicyclononan-9-ol 4-chlorobenzoate ester;
3,7-di(4-(3,4-dimethoxyphenyl)butyl)-3,7-diazabicyclononan-9-ol 4-chlorobenzoate ester;
3,7-di(furanylmethyl)-3,7-diazabicyclononan-9-ol 4-chlorobenzoate ester;
3,7-di(4-(3,4-ethylenedioxyphenyl)butyl)-3,7-diazabicyclononan-9-ol 4-chlorobenzoate ester;
Syn-7-(4-(3,4-methylenedioxyphenyl)butyl)-3-oxa-7-azabicyclo[3.3.1]nonan-9-ol 4-chlorobenzoate ester;
Syn-7-hexyl-3-oxa-7-azabicyclo[3.3.1]nonan-9-ol 4-chlorobenzoate ester;
Syn-7-(4-(3,4-methylenedioxyphenyl)butyl)-3-oxa-7-azabicyclo[3.3.1]nonan-9-ol 4-chlorobenzyl ether;
Syn-7-hexyl-3-oxa-7-azabicyclo[3.3.1]nonan-9-ol 4-chlorobenzyl ether;
syn-7-(2-benzyloxypropyl)-3-thia-7-azabicyclo[3.3.1]nonan-9-ol 4-chlorobenzoate ester;
and pharmaceutically acceptable salts or solvates thereof.
A particularly preferred compound is a compound of formula (1a) (Example 1) of a pharmaceutically acceptable salt or solvate thereof. 
Those skilled in the art will recognize that stereocenters exist in the compounds of Formula (1). Accordingly, the present invention includes all possible stereoisomers and geometric isomers of Formula (1) and includes not only racemic compounds but also the optically active isomers as well. When a compound of Formula (1) is desired as a single enantiomer, it may be obtained either by resolution of the final product or by stereospecific synthesis from either isomerically pure starting material or any convenient intermediate. Resolution of the final product, an intermediate or a starting material may be effected by any suitable method known in the art. See, for example, Stereochemistry of Carbon Compounds by E. L. Eliel (Mcgraw Hill, 1962) and Tables of Resolving Agents by S. H. Wilen. Additionally, in situations where tautomers of the compounds of Formula (1) are possible, the present invention is intended to include all tautomeric forms of the compounds.
It will also be appreciated by those skilled in the art that the compounds of the present invention may also be utilized in the form of a pharmaceutically acceptable salt or solvate thereof. The physiologically acceptable salts of the compounds of Formula (1) include conventional salts formed from pharmaceutically acceptable inorganic or organic acids or bases as well as quaternary ammonium acid addition salts. More specific examples of suitable acid salts include hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric, citric, pamoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric, toluenesulfonic, methanesulfonic, naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic, malic, steroic, tannic and the like. Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable salts. More specific examples of suitable basic salts include sodium, lithium, potassium, magnesium, aluminium, calcium, zinc, N,N1-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine salts. References hereinafter to a compound according to the invention include both compounds of Formula (I) and their pharmaceutically acceptable salts and solvates.
It will be appreciated by those skilled in the art that reference herein to treatment extends to prophylaxis as well as the treatment of established diseases or symptoms. Moreover, it will be appreciated that the amount of a Kv2.1 antagonist required for use in treatment will vary with the nature of the condition being treated and the age and the condition of the patient and will be ultimately at the discretion of the attendant physician or veterinarian. In general, however, doses employed for adult human treatment will typically be in the range of 0.02-5000 mg per day, e.g., 1-1500 mg per day. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day.
While it is possible that the Kv2.1 antagonist, e.g. a compound of Formula 1 may be therapeutically administered as the raw chemical, it is preferable to present the active ingredient as a pharmaceutical formulation. Accordingly, the present invention further provides for a pharmaceutical formulation comprising a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof together with one or more pharmaceutically acceptable carriers therefor and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. There is further provided by the present invention a process of preparing a pharmaceutical formulation comprising a compound of formula (I), which process comprises admixing a compound of formula (I), or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers therefor, and optionally other therapeutic and/or prophylactic ingredients.
Formulations comprising Kv2.1 antagonists of the present invention include those especially formulated for oral, buccal, parenteral, transdermal, inhalation, intranasal, transmucosal, implant, or rectal administration, however, oral administration is preferred. For buccal administration, the formulation may take the form of tablets or lozenges formulated in a conventional manner. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, (for example, syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch or polyvinylpyrrolidone), fillers (for example, lactose, sugar, microcrystalline cellulose, maize-starch, calcium phosphate or sorbitol), lubricants (for example, magnesium stearate, stearic acid, talc, polyethylene glycol or silica), disintegrants (for example, potato starch or sodium starch glycollate) or wetting agents, such as sodium lauryl sulfate. The tablets may be coated according to methods well-known in the art.
Alternatively, Kv2.1 antagonists may be incorporated into oral liquid preparations such as aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, for example. Moreover, formulations containing these compounds may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents such as sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible fats; emulsifying agents such as lecithin, sorbitan mono-oleate or acacia; non-aqueous vehicles (which may include edible oils) such as almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; and preservatives such as methyl or propyl p-hydroxybenzoates or sorbic acid. Such preparations may also be formulated as suppositories, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
Additionally, Kv2.1 antagonists may be formulated for parenteral administration by injection or continuous infusion. Formulations for injection may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before use.
The formulations according to the invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Accordingly, the compounds of the invention may be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins or as sparingly soluble derivatives as a sparingly soluble salt, for example.
The formulations according to the invention may contain between 0.1-99% of the active ingredient, conveniently from 30-95% for tablets and capsules and 3-50% for liquid preparations.
The present invention also provides a method of identifying extrinsic materials which possess the ability to modulate Kv2.1 channel activity and thereby modify insulin secretion.
For example the invention provides a method of detecting blockers of the Kv2.1 potassium channel which comprises measuring changes in intracellular Ca2+ in a suitable cell line in the presence of glucose and in the additional absence or presence of a compound which may have antagonist activity. For example, xcex2TC3-neo insulinoma cells respond to a change in glucose with a rise in intracellular Ca2+. Their glucose sensitivity is markedly different from normal pancreatic xcex2-cells; a maximal response is produced by 1 mM glucose whereas normal xcex2-cells are sensitive to glucose in the range 6-30 mM. This cell line represents an appropriate model system to study delayed rectifier potassium channels since they express Kv2.1 channels, and respond to nonselective blockers of these channels (like TEA) with a glucose dependent rise in intracellular C2+. Other assay modalities are possible to detect blockers of delayed receifier potassium channels in xcex2TC3-neo cells. These include monitoring changes in membrane potential with a voltage-sensitive dye (e.g. bis-oxonol) or measure changes in intracellular K+ with a K+-sensitive dye (e.g. PBFI).
Additionally, the invention provides a method of expressing Kv2.1 in a suitable cell line, for example, Chinese Hamster Ovary (CHO) cells. This cell line may be treated with compounds to measure their ability to modulate Kv2.1 activity e.g. by measuring change in K+ current, e.g. the standard whole cell patch clamp methods described in the Examples section hereinafter.
Other agents have been postulated to exert glucose sensitive secretagogue activity, for example glucagon-like peptide 1 (GLP-1). This peptide has been reported to potentiate glucose-induced insulin secretion in part by elevating intracellular Ca2+ Accordingly the invention further provides a method for assaying for compounds which display glucose sensitive secretagague activity which comprises measuring changes in intracellular Ca2+ in a suitable cell line in the presence of glucose and in the additional presence or absence of a compound which may have glucose sensitive secretagague activity. An alternative method for measuring the activity of antagonists of Kv2.1 to enhance insulin secretion is the perfused pancreas method. This is described in more detail in the Examples.
An alternative method for detecting unknown molecules with activity at Kv2.1 delayed rectifier potassium channels involving measurement of displacement of labeled hanatoxin (purified from natural sources or recombinant or synthetic). For instance, there are several tyrosine residues in hanatoxin that could be conveniently radiolabelled, and the resulting radiolabeled hanatoxin could then be employed in a binding assay.
The compounds of the present invention may be prepared according to the following general synthetic schemes. It will be appreciated that a skilled person would readilly adapt the schemes to prepare a particular compound of formula (I).
The synthesis of compounds of formula 1 in which
R is as defined above,
R1 is 4-chlorobenzoyl,
R3 is H,
and X is Nxe2x80x94R2 where R2 is methyl is outlined in scheme 1.
The ketone, 7-methyl-3-benzyl-3,7-diazabicyclononan-9-one, was reduced with lithium aluminum hydride and the benzyl group of the resulting alcohol was removed under hydrogenation conditions. The resultant free amine was protected with a BOC group and the alcohol was converted to the ester by treatment with an acetylating agent, such as 4-chlorobenzoyl chloride to give anti-3-(tert-butoxycarbonyl)-7-methyl-3,7-diazabicyclononan-9-ol 4-chlorobenzoate ester. Separation of the geometric isomers obtained from the lithium aluminum hydride reduction can be carried out at several points along the synthetic pathway. In this case the isomers were separated after the introduction of the ester group. The stereochemistry of anti-3-(tert-butoxycarbonyl)-7-methyl-3,7-diazabicyclononan-9-ol 4-chlorobenzoate ester was determined by NMR nOe and X-ray crystallographic analysis. Removal of the BOC group was accomplished by treatment with a strong acid such as trifluoroacetic acid, and the resulting secondary amine was reacted with an aldehyde in the presence of a reducing agent such as sodium triacetoxyborohydride to give the desired compound. Compounds of formula 1 can be converted to an acid addition salt such as the HCl salt by simple treatment with HCl in an organic solvent. 
To 3-methyl,7-benzyl-3,7-diazabicyclo[3,3,1]nonan-9-one in dry tetrahydrofuran (350 mL) was added lithium aluminum hydride (41 mL, 1M in tetrahydrofuran) dropwise. The mixture was stirred for 30 min and sodium hydroxide (1M, aqueous) was add to consume excess lithium aluminum hydride. The mixture was filtered and the solvent was removed under reduced pressure. The resultant oily residue was dissolved in ethanol water (1:1, 200 mL) and the pH was adjusted to 1 with HCl (conc). Palladium on activated carbon (10%, 250 mg) was added and the mixture was hydrogenated (50 psi) for 12 h. The mixture was filtered and the solvent removed under reduced pressure. To the resulting light yellow residue, dissolved in chloroform (400 mL) and triethylamine (10 mL), was added 4-dimethylaminopyridine (4.88 g, 0.04 mol) and di-tert-butyl dicarbonate (8.72 g; 0.04 mol). The mixture was stirred for 4 h and 4-chlorobenzoyl chloride (7.7 g, 0.04 mol) was added. The mixture was stirred overnight and washed with sodium bisulfate (1M, aqueous), water and sodium bicarbonate (saturated aqueous). The organic phase was dried with magnesium sulfate and solvent evaporated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with 80:20 hexane:ethyl acetate to give the title compound (6 g, 37%) as a white solid: 1H NMR (400 MHz, CDCl3) 7.99 (d, J=8.5Hz, 2H), 7.44 (d, J=8.5Hz, 2H), 5.10 (m, 1H), 4.52 (d, J=13.2 Hz, 1H), J=13.2 Hz, 1H), 3.20 (d, J=13.2 Hz, 1H), 3.07 (d, J=13.2 Hz, 1H), 3.00-2.50 (m, 4H), 2.15 (s, 3H), 2.05-1.96 (m, 2H), 1.47 (s, 9H); mass spectrum m/z 395 (M+H)+.